1. Field of the Invention
This invention relates to the immobilization of immunoglobulins, and particularly the immobilization of IgG's by binding to protein A. Such binding presents a range of utility in immunological techniques, both analytical and preparatory, and is of particular interest in the purification of antibodies, notably monoclonal, from an ascites fluid.
2. Description of the Prior Art
The ability of protein A to bind to the Fc portion of IgG molecules is widely used as a basis for separation of IgG's from other proteins by affinity chromatography. In such separations, protein A is generally immobilized by cross-linking to a solid phase support such as agarose, and the sample is applied as a solution in a buffer which disfavors the binding of other proteins in the mixture. The immunoglobulins are then recovered from the solid phase by elution using buffers of altered composition. The separation achieved, however, is less than complete.
An early disclosure of the binding of certain antibodies to protein A is found in Kronvall et al., Journal of Immunology, 105(5): 1116 (1970). An attempt to improve the separation was made by Mackenzie, et al., Journal of Immunology, 120(5): 1493 (1978), in which a continuously increasing NaSCN gradient at low pH was used as an elution buffer with a protein A-Sepharose 4B column. Binding was low for IgG.sub.1 and IgG.sub.2 and reproducibility has been found to be lacking.
An elution sequence characterized by a stepwise decrease in pH at constant low salt concentration was used by Ey et al., Immunology, 15: 429 (1978). A substantial amount of IgG.sub.1 was found to have bled over into the subsequent fraction, and reproducibility was lacking here also.
Chalon et al., in Scand. Journal of Immunology, 9: 359 (1979), achieved partial success in separating various IgG's from IgA and IgM, by dissolving the mixtures in phosphate-buffered saline at pH 7.3, then introducing the solution into a column containing protein A-Sepharose 4B equilibrated to the same buffer. Two peaks emerged with no change in buffer, the second of which to elute containing mostly IgG.sub.1. The yields varied from 31% to 73%.
Finally, sixteen different eluents for protein A-Sepharose 4B purification of various IgG's were studied by Bywater et al., Journal of Immunological Methods, 64: 1 (1983). No improvement in either separation or yield over previous methods was observed in any of the samples tested.